Modulation of Neutrophil Function by a Secreted Mucinase of Escherichia coli O157∶H7

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Abstract
Escherichia coli O157∶H7 is a human enteric pathogen that causes hemorrhagic colitis which can progress to hemolytic uremic syndrome, a severe kidney disease with immune involvement. During infection, E. coli O157∶H7 secretes StcE, a metalloprotease that promotes the formation of attaching and effacing lesions and inhibits the complement cascade via cleavage of mucin-type glycoproteins. We found that StcE cleaved the mucin-like, immune cell-restricted glycoproteins CD43 and CD45 on the neutrophil surface and altered neutrophil function. Treatment of human neutrophils with StcE led to increased respiratory burst production and increased cell adhesion. StcE-treated neutrophils exhibited an elongated morphology with defective rear detachment and impaired migration, suggesting that removal of the anti-adhesive capability of CD43 by StcE impairs rear release. Use of zebrafish embryos to model neutrophil migration revealed that StcE induced neutrophil retention in the fin after tissue wounding, suggesting that StcE modulates neutrophil-mediated inflammation in vivo. Neutrophils are crucial innate effectors of the antibacterial immune response and can contribute to severe complications caused by infection with E. coli O157∶H7. Our data suggest that the StcE mucinase can play an immunomodulatory role by directly altering neutrophil function during infection. StcE may contribute to inflammation and tissue destruction by mediating inappropriate neutrophil adhesion and activation. Enterohemorrhagic Escherichia coli (EHEC) poses a significant threat to the U.S. food supply, causing foodborne gastrointestinal disease in humans that can progress to hemolytic uremic syndrome (HUS), a potentially fatal kidney disease. Research suggests that EHEC strains are growing more virulent, resulting in a higher incidence of hospitalization and development of HUS from recent produce-associated outbreaks. Although immune dysregulation is a feature of HUS disease, the specific mechanisms contributing to altered immune function require investigation. Furthermore, the contribution of the immune response to early intestinal disease is not known. StcE is a secreted protease of EHEC that is expressed during infection and may contribute to virulence via cleavage of mucin-like glycoproteins. In this study, we define mucinase activity toward glycoproteins on the surface of human neutrophils and find that StcE alters neutrophil activity by interacting with these proteins. StcE affected crucial neutrophil functions including oxidative burst production and migration. The effects of StcE were both cleavage-dependent and cleavage-independent, providing insight into a novel mechanism for mediating neutrophil function via mucin interactions. Our study reports an immune-modulating role for a potential EHEC virulence factor and provides a possible explanation for altered neutrophil phenotypes observed during E. coli O157∶H7-induced disease.